A mosaic tetracycline resistance gene tet(S/M) detected in an MDR pneumococcal CC230 lineage that underwent capsular switching in South Africa.

OBJECTIVES: We reported tet(S/M) in Streptococcus pneumoniae and investigated its temporal spread in relation to nationwide clinical interventions. METHODS: We whole-genome sequenced 12 254 pneumococcal isolates from 29 countries on an Illumina HiSeq sequencer. Serotype, multilocus ST and antibiotic resistance were inferred from genomes. An SNP tree was built using Gubbins. Temporal spread was reconstructed using a birth-death model. RESULTS: We identified tet(S/M) in 131 pneumococcal isolates and none carried other known tet genes. Tetracycline susceptibility testing results were available for 121 tet(S/M)-positive isolates and all were resistant. A majority (74%) of tet(S/M)-positive isolates were from South Africa and caused invasive diseases among young children (59% HIV positive, where HIV status was available). All but two tet(S/M)-positive isolates belonged to clonal complex (CC) 230. A global phylogeny of CC230 (n=389) revealed that tet(S/M)-positive isolates formed a sublineage predicted to exhibit resistance to penicillin, co-trimoxazole, erythromycin and tetracycline. The birth-death model detected an unrecognized outbreak of this sublineage in South Africa between 2000 and 2004 with expected secondary infections (effective reproductive number, R) of ∼2.5. R declined to ∼1.0 in 2005 and <1.0 in 2012. The declining epidemic could be related to improved access to ART in 2004 and introduction of pneumococcal conjugate vaccine (PCV) in 2009. Capsular switching from vaccine serotype 14 to non-vaccine serotype 23A was observed within the sublineage. CONCLUSIONS: The prevalence of tet(S/M) in pneumococci was low and its dissemination was due to an unrecognized outbreak of CC230 in South Africa. Capsular switching in this MDR sublineage highlighted its potential to continue to cause disease in the post-PCV13 era.

Helicobacter pylori genotypes among Belarus patients with gastroduodenal disorders and their association with clinical outcome.

The aim of this study was to evaluate the prevalence of Helicobacter pylori genotypes (vacA and cagPAI) directly in gastric biopsy specimens in patients with gastric diseases in Belarus. Gastric biopsies were collected from 461 patients with different gastrointestinal disorders: superficial gastritis (287 subjects), atrophy gastritis (59 subjects), erosive gastritis (47 subjects), duodenal ulcer disease (54 subjects), and stomach ulcer (14 subjects). PCR-based genotyping was used to detect s1a, s1b, s2, m1a, m1b, m2, cagM, cagA, and cagT genes. Overall prevalence of vacA s1a allele was 60.5% followed by m2 (47.1%) and m1a (37.5%). The analysis of data showed that genotype s1a/m1a was significantly more prevalent in patients with duodenal ulcer (21.4% vs. 45.1%, OR = 3.0, 95% CI = 1.5-6.1). The cagA gene was found with a high incidence in most patients with inflammatory diseases of stomach and duodenum. There was a significant increase in the frequency of cagT in patients with duodenal ulcer as compared to superficial gastritis. A high cagM prevalence was found in patients with atrophy gastritis and duodenal ulcer disease. All three island genes of pathogenicity of cagPAI are more often detected in patients with duodenal ulcer, which increases the risk of developing duodenal ulcer by 4.5 times.

Serogroup diversity and antibiotic susceptibility of Neisseria meningitidis: Meningococcus infection monitoring in Belarus.

This study performed an epidemiological survey of Neisseria meningitidis strains isolated from patients and from asymptomatic carriers. Altogether, 74 N. meningitidis strains (46 invasive and 28 non-invasive) were isolated between February 2011 and May 2018 in different regions of the Republic of Belarus. Serogenotyping was carried out by real-time PCR. Minimum inhibitory concentrations (MICs) of antibiotics were determined by broth microdilution and results were interpreted in accordance with EUCAST. The serogroups of N. meningitidis were determined as follows: serogroup B - 65%, C - 11%, W - 9%, A - 5%, Y - 4%, and Z and NG - 3% each. The MIC50 and MIC90 for benzylpenicillin (0.032/0.064-0.125 mg/L), ampicillin (0.032/0.125 mg/L), amoxicillin (0.125/0.25 mg/L), cefotaxime (0.016/0.016 mg/L), ceftriaxone (0.002/0.016 mg/L), ciprofloxacin (0.004/0.008 mg/L), chloramphenicol (1/1 mg/L), meropenem (0.008/0.008-0.016 mg/L), tetracycline (0.25/0.5 mg/L), and rifampicin (0.016/0.25 mg/L) were established. Strains with intermediate susceptibility for benzylpenicillin (12.3%), ampicillin (6.8%), and amoxicillin (24.7%) have been identified. In this study, we report the first rifampicin-resistant N. meningitidis in Belarus.

Genomic analysis of endemic clones of toxigenic and non-toxigenic Corynebacterium diphtheriae in Belarus during and after the major epidemic in 1990s.

BACKGROUND: Diphtheria remains a major public health concern with multiple recent outbreaks around the world. Moreover, invasive non-toxigenic strains have emerged globally causing severe infections. A diphtheria epidemic in the former Soviet Union in the 1990s resulted in ~5000 deaths. In this study, we analysed the genome sequences of a collection of 93 C. diphtheriae strains collected during and after this outbreak (1996 - 2014) in a former Soviet State, Belarus to understand the evolutionary dynamics and virulence capacities of these strains. RESULTS: C. diphtheriae strains from Belarus belong to ten sequence types (STs). Two major clones, non-toxigenic ST5 and toxigenic ST8, encompassed 76% of the isolates that are associated with sore throat and diphtheria in patients, respectively. Core genomic diversity is limited within outbreak-associated ST8 with relatively higher mutation rates (8.9 × 10-7 substitutions per strain per year) than ST5 (5.6 × 10-7 substitutions per strain per year) where most of the diversity was introduced by recombination. A variation in the virulence gene repertoire including the presence of tox gene is likely responsible for pathogenic differences between different strains. However, strains with similar virulence potential can cause disease in some individuals and remain asymptomatic in others. Eight synonymous single nucleotide polymorphisms were observed between the tox genes of the vaccine strain PW8 and other toxigenic strains of ST8, ST25, ST28, ST41 and non-toxigenic tox gene-bearing (NTTB) ST40 strains. A single nucleotide deletion at position 52 in the tox gene resulted in the frameshift in ST40 isolates, converting them into NTTB strains. CONCLUSIONS: Non-toxigenic C. diphtheriae ST5 and toxigenic ST8 strains have been endemic in Belarus both during and after the epidemic in 1990s. A high vaccine coverage has effectively controlled diphtheria in Belarus; however, non-toxigenic strains continue to circulate in the population. Recombination is an important evolutionary force in shaping the genomic diversity in C. diphtheriae. However, the relative role of recombination and mutations in diversification varies between different clones.

Antimicrobial susceptibility/resistance and NG-MAST characterisation of Neisseria gonorrhoeae in Belarus, Eastern Europe, 2010-2013.

BACKGROUND: Gonorrhoea and widely spread antimicrobial resistance (AMR) in its etiological agent Neisseria gonorrhoeae are major public health concerns worldwide. Gonococcal AMR surveillance nationally and internationally, to identify emerging resistance and inform treatment guidelines, is imperative for public health purposes. In 2009, AMR surveillance was initiated in Belarus, Eastern Europe because no gonococcal AMR data had been available for at least two decades. Herein, the prevalence and trends of gonococcal AMR and molecular epidemiological characteristics of N. gonorrhoeae strains from 2010 to 2013 in Belarus, are described. METHODS: N. gonorrhoeae isolates (n=193) obtained in the Mogilev (n=142), Minsk (n=36) and Vitebsk (n=15) regions of Belarus in 2010 (n=72), 2011 (n=6), 2012 (n=75) and 2013 (n=40) were analyzed in regards to AMR using the Etest method and for molecular epidemiology with N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: During 2010-2013, the proportions of resistant N. gonorrhoeae isolates were as follows: tetracycline 36%, ciprofloxacin 28%, penicillin G 9%, azithromycin 5%, and cefixime 0.5%. Only one (0.5%) ß-lactamase producing isolate was detected. No isolates resistant to ceftriaxone and spectinomycin were identified. Overall, the resistance levels to tetracycline, ciprofloxacin and penicillin G were relatively stable. Interestingly, the level of resistance to azithromycin declined from 12% in 2010 to 0% in 2013 (P < 0.05). In total, 70 NG-MAST STs were identified. The predominant STs were ST1993 (n=53), ST807 (n=13), ST285 (n=8) and ST9735 (n=8). Many novel STs (n=43, 61%), representing 41% of all isolates, were found. CONCLUSIONS: During 2010-2013, the N. gonorrhoeae population in Belarus displayed high and relatively stable resistance levels to tetracycline, ciprofloxacin, and penicillin G, while the resistance to azithromycin declined. One isolate was resistant to cefixime, but no resistance to ceftriaxone or spectinomycin was found. The results of the present surveillance initiated in 2009 were also used to replace penicillin G with ceftriaxone (1 g single dose intramuscularly) as the first-line drug for empiric treatment of gonorrhoea in the national treatment guidelines in Belarus in late 2009. It is essential to further strengthen the surveillance of gonococcal AMR and ideally survey also treatment failures and molecular epidemiological genotypes in Belarus.

Determination of principal genotypic groups among susceptible, MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and Iran.

INTRODUCTION: All members of the Mycobacterium tuberculosis complex were assigned to one of the three principle genetic groups based on KatG463/GyrA95 polymorphism. MATERIALS AND METHODS: A total of 202 isolates of M. tuberculosis consisting of 50 susceptible, 121 MDR (multidrug resistant) and 31 XDR (extensively drug resistant) isolated from culture-confirmed tuberculosis patients in different regions of Belarus and Iran (Tehran and Markazi province). Isolates were screened by sequencing and polymerase chain reaction restriction fragment length polymorphism (RFLP) assay, and were further divided into three principal genetic groups (PGG), based on Sreevatsan's pattern as polymorphisms in KatG463/GyrA95 codons. RESULTS: Among the 104 isolates, characterized as MDR from Belarus, 57 (54.8 ± 4.8%), 30 (28.8 ± 4.43%), 17 (16.3 ± 3.6), belonged to PGG 1, 2, and 3, respectively (p< 0.05). Thirty one XDR isolates from Belarus had a similar pattern as 15 (48.4%), 12 (38.7%), 4 (12.9%) PGG 1, 2, and 3, respectively. From Iranian samples, Markazi isolates (susceptible to drugs) had a pattern as 12 (36.5%), 15 (45.5%), 3 (6%), and Tehran samples were (selected MDR): 9 (53%), 6 (35.2%), 2 (11.8%) (PGG 1, 2, and 3, respectively). In a study of tuberculosis patients, who were in prison, no relation was found between PGG and resistance to isoniazid, but most of the identified isolates belonged to PGG 1 (45.5 ± 10.9%) (p< 0.05). Overall, the group 1 isolates showed more frequency in MDR and XDR rather than susceptible strains, and there aren't any relations to geographic region.

A Streptococcus pneumoniae lineage usually associated with pneumococcal conjugate vaccine (PCV) serotypes is the most common cause of serotype 35B invasive disease in South Africa, following routine use of PCV.

Pneumococcal serotype 35B is an important non-conjugate vaccine (non-PCV) serotype. Its continued emergence, post-PCV7 in the USA, was associated with expansion of a pre-existing 35B clone (clonal complex [CC] 558) along with post-PCV13 emergence of a non-35B clone previously associated with PCV serotypes (CC156). This study describes lineages circulating among 35B isolates in South Africa before and after PCV introduction. We also compared 35B isolates belonging to a predominant 35B lineage in South Africa (GPSC5), with isolates belonging to the same lineage in other parts of the world. Serotype 35B isolates that caused invasive pneumococcal disease in South Africa in 2005-2014 were characterized by whole-genome sequencing (WGS). Multi-locus sequence types and global pneumococcal sequence clusters (GPSCs) were derived from WGS data of 63 35B isolates obtained in 2005-2014. A total of 262 isolates that belong to GPSC5 (115 isolates from South Africa and 147 from other countries) that were sequenced as part of the global pneumococcal sequencing (GPS) project were included for comparison. Serotype 35B isolates from South Africa were differentiated into seven GPSCs and GPSC5 was most common (49 %, 31/63). While 35B was the most common serotype among GPSC5/CC172 isolates in South Africa during the PCV13 period (66 %, 29/44), 23F was the most common serotype during both the pre-PCV (80 %, 37/46) and PCV7 period (32 %, 8/25). Serotype 35B represented 15 % (40/262) of GPSC5 isolates within the global GPS database and 75 % (31/40) were from South Africa. The predominance of the GPSC5 lineage within non-vaccine serotype 35B, is possibly unique to South Africa and warrants further molecular surveillance of pneumococci.

High level isoniazid resistance correlates with multiple mutation in the katG encoding catalase proxidase of pulmonary tuberculosis isolates from the frontier localities of Iran.

The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Iran. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG gene amplification, and DNA sequencing analysis were performed. Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG gene. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC-->ACC), 316 (GGC-->AGC), 309 (GGT-->GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC ≥ 5-10 µg/mL). Furthermore, it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively. In this study, 62% (n= 21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC-->ACC), 316 (GGC-->GTT), 309 (GGT-->GGT), and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5-10 µg/mL).

RETRACTED: High level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran.

This article has been retracted.

High-level rifampin resistance correlates with multiple mutations in the rpoB gene of pulmonary tuberculosis isolates from the Afghanistan border of Iran.

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (beta-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG-->GCG), 531 (TCG-->TTG or TTC), and 526 (CAC-->CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, >or=100 microg/ml).

Application of Molecular Methods for Carbapenemase Detection.

This study has evaluated the correlation between different carbapenemases detection methods on carbapenem non-susceptible Klebsiella pneumoniae strains from Northern and Eastern Europe; 31 institutions in 9 countries participated in the research project, namely Finland, Estonia, Latvia, Lithuania, Russia, St. Petersburg, Poland, Belarus, Ukraine, and Georgia. During the research program, a total of 5,001 clinical K. pneumoniae isolates were screened for any carbapenem non-susceptibility by the disk diffusion method, Vitek 2 or Phoenix system following the EUCAST guideline on detection of resistance mechanisms, version 1.0. Strains isolated from outpatients and hospitalized patients from April 2015 to June 2015 were included. All types of samples (blood, pus, urine, etc.) excluding fecal screening or fecal colonization samples have been represented. In total, 171 carbapenemase screening-positive K. pneumoniae isolates (3.42%) were found and characterized. Several methods were used for detection of carbapenemases production, including Luminex assay (PCR and hybridization), whole genome sequencing, MALDI-TOF based Imipenem degradation assay, and immunochromatography testing. Minimal inhibitory concentration determination for Meropenem by agar-based gradient method was also used. Finally, 83 K. pneumoniae strains were carbapenemase negative by all confirmation methods (49.4% of all screening-positive ones), 74 - positive by three methods (44.0%), 8 - positive by two methods (4.8%) and 3 - positive by only one method (1.8%). The sensitivity of the tests was 96.3% for Whole genome sequencing and MALDI-TOF assay (both three undetected cases), and 95.1% for Luminex-Carba (4 undetected cases). The most commonly detected carbapenemases were NDM (n = 54) and OXA-48 (n = 26), followed by KPC-2, VIM-5, and OXA-72 (one case of each). Our results showed that different types of carbapenemases can be detected in the countries involved in the project. The sensitivity of our methods for carbapenemase detection (including screening as a first step and further confirmation tests) was >95%, but we would recommend using different methods to increase the sensitivity of detection and make it more precise.

Prevention and control of meningococcal disease: Updates from the Global Meningococcal Initiative in Eastern Europe.

The Global Meningococcal Initiative (GMI) aims to prevent invasive meningococcal disease (IMD) worldwide through education, research and cooperation. In March 2019, a GMI meeting was held with a multidisciplinary group of experts and representatives from countries within Eastern Europe. Across the countries represented, IMD surveillance is largely in place, with incidence declining in recent decades and now generally at <1 case per 100,000 persons per year. Predominating serogroups are B and C, followed by A, and cases attributable to serogroups W, X and Y are emerging. Available vaccines differ between countries, are generally not included in immunization programs and provided to high-risk groups only. Available vaccines include both conjugate and polysaccharide vaccines; however, current data and GMI recommendations advocate the use of conjugate vaccines, where possible, due to the ability to interrupt the acquisition of carriage. Ongoing carriage studies are expected to inform vaccine effectiveness and immunization schedules. Additionally, IMD prevention and control should be guided by monitoring outbreak progression and the emergence and international spread of strains and antibiotic resistance through use of genomic analyses and implementation of World Health Organization initiatives. Protection of high-risk groups (such as those with complement deficiencies, laboratory workers, migrants and refugees) is recommended.

Frequency and molecular characterization of isoniazid resistance in katG region of MDR isolates from tuberculosis patients in southern endemic border of Iran.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis collected from patients in southern endemic region of Iran. Drug susceptibility testing was determined by using the BACTEC system and CDC standard conventional proportional method. All 28 isolates were resistant to isoniazid (100%), rifampicine (14%) streptomycin (82%) and 8 (28%) was resistant to ethambutol. Mono-resistance to isoniazid was observed in four isolates (14%). In 21 isolates three types of mutations were found in codon 315: AGC-->ACC (Ser-->Thr) (61%), AGC-->AGG (Ser-->Arg) (3.5%) and AGC-->AAC (Ser-->Asn) (11%). Moreover, one type of mutation was seen in codons 311 (32%), 299 (28.5%), and 322 (21.5%). Of 28 isolates 12 (46%) found with one mutation, 7 (27%) had two, 5 (19%) had three and 2 (8%) revealed to have four mutations in katG gene. In nine isolates single mutation was observed in codon 311 (GAC-->TAC). This study indicates presence of multiple mutations in codon 315 and 299 among multiple drug-resistant (MDR) strains of M. tuberculosis collected from patients sputum having secondary infections in adjacent southern endemic border of Iran.

Identification of mutations in the rpoB encoding the RNA polymerase beta subunit in rifampicine-resistant Mycobacterium tuberculosis strains from Iran.

The aim of this study was to investigate the frequency, location and type of rpoB mutations in Mycobacterium tuberculosis isolated from patients in Iran. 91 sputum were collected from suspected tuberculosis patients, 34 Rif-r isolates (87%) were identified as M. tuberculosis. Polymerase chain reaction (PCR) amplification and DNA sequencing methods were performed. 411 bp fragments of rpoB gene were sequenced and mutations in 81 bp regions were analyzed. 60 mutations and 13 micro deletions were identified in 29 RIF-r MBT (85%). Among 60 mutations, 6 silent and 54 missense were identified. Missense mutations produced 23 types of amino acid substitutions. In 5 RIF-r MBT isolates (15%) no mutations were found in the core region of the rpoB gene. All silent mutations were localized in codon 507. Most frequent mutations detected from Iranian strains were in codons 523 and 526. Five alleles in codon 526 and 3 alleles in triplets in each codons 507, 508, 513 were found. 6 (19%) strains harboured single mutations 6 (18%) placed in codons 526, 510 while the rest of isolates 23 (69%) had multiple mutations: Double 11 (34%), triple 7 (22%), and quartile mutations 1 (3%) and 4 (12%) of strains harboured 5 mutations respectively.

katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis isolates from Belarusian patients.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Mutations in different codons causing resistance to isoniazide in the gene of catalase peroxides (katG) in Belarusian strains was determined. 42 strains of Rif-r and Inhr(MDR) were isolated in different regions of Belarus. Culture susceptibility testing of all 42 strains revealed resistance to streptomycin (90%), 16 strains (43%) were resistant to etambutol. DNA Extraction, Standard PCR identification and katG gene amplification were performed. The most affected codons of katG gene were 315(95%), 316(16.2%), and 309(14.5%). Four types of mutations were identified in codon 315: AGC-->ACC (n=36)32.4%, AGC-->AGG (n=1) 0.9%, AGC-->AAC (n=2) 1.8%, AGC-->GGC (n=1) 0.9%. One type of mutation was found in codon 316: GGC-->AGC (n=18)16.2%, four types of mutations were detected in codon 309: GGT-->GGT (n=7)6.3%, GGT-->GCT (n=4)3.6%, GGT-->GTC (n=3)2.7%, GGT-->GGG (n=1)0.9%. Mutations in codon 309 make up 34%, in codon 316 (37%) and other types of mutations 29% of all detected mutations. In 2 isolated strains mutation were identified in codons 463, 35 and, in codons 454, 357 respectively and 2 isolates, there were not found any mutations. Concluding, all INH-r MBT had resistance-associated nucleotide changes mostly in codons 315.

Transmission Electron Microscopy of XDR Mycobacterium tuberculosis Isolates Grown on High Dose of Ofloxacin.

The aim of the study was to investigate behavior of resistant Mycobacterium tuberculosis (MTB) isolates under a high dose of ofloxacin and its morphological changes. 19 extensively drug resistant (XDR) clinical isolates of MTB were grown on Löwenstein-Jensen medium containing progressively increasing concentrations of ofloxacin (2, 4, 8, 16, 32 mg/L). Ultra-structure analyses of resistant isolates grown on ofloxacin were conducted by transmission electron microscopy (TEM). Fixation was carried out by 4% glutaraldehyde in 0.1 M sodium cacodylate buffer on 300 mesh carbon formvar copper grid. The samples were negatively stained with uranium acetate suspension. All19XDRMTBisolatesweregrownandformedcoloniessuccessfullyon2,4,8mg/L,sevenisolates on16mg/L,andfourisolateson32mg/Lofloxacin. Morphologicalchangesandunusualformswere detected in 8, 16 and 32 mg/L ofloxacin at 43%, 76.5% and 81% of cells, respectively. Swollen form (protoplast like), ghost-like cell, degraded forms, and in a few cases, detached cytoplasm from cell wall were clearly detected in high drug concentrations in comparison to control. Changes in morphology were increased with increasing ofloxacin concentrations (p < 0.05). Some XDR isolates could be successfully grown on high doses of ofloxacin (32 mg/L), but with changes in morphology. It was concluded that several magnitudes of the drug doses could not prevent growth of drug resistant forms.

Molecular epidemiology of C. diphtheriae strains during different phases of the diphtheria epidemic in Belarus.

BACKGROUND: The reemergence of epidemic diphtheria in Belarus in 1990s has provided us with important information on the biology of the disease and the diversity of the causative agent Corynebacterium diphtheriae. Molecular investigations were conducted with the aim to analyze the genetic variability of C diphtheriae during the post-epidemic period. METHODS: The biotype and toxigenicity status of 3513 C. diphtheriae strains isolated from all areas in Belarus during a declining period of diphtheria morbidity (1996-2005) was undertaken. Of these, 384 strains were isolated from diphtheria cases, 1968 from tonsillitis patients, 426 from contacts and 735 from healthy carriers. Four hundred and thirty two selected strains were ribotyped. RESULTS: The C diphtheriae gravis biotype, which was prevalent during 1996-2000, was "replaced" by the mitis biotype during 2001-2005. The distribution of toxigenic C. diphtheriae strains also decreased from 47.1% (1996) to 5.8% (2005). Changes in the distribution of the epidemic ribotypes Sankt-Peterburg and Rossija were also observed. During 2001-2005 the proportion of the Sankt-Peterburg ribotype decreased from 24.3% to 2.3%, in contrast to the Rossija ribotype, that increased from 25.1% to 49.1%. The circulation of other toxigenic ribotypes (Otchakov, Lyon, Bangladesh), which were prevalent during the period of high diphtheria incidence, also decreased. But at the same time, the proportion of non-toxigenic strains with the Cluj and Rossija ribotypes dramatically increased and accounted for 49.3% and 30.1%, respectively. CONCLUSION: The decrease in morbidity correlated with the dramatic decrease in the isolation of the gravis biotype and Sankt Peterburg ribotype, and the prevalence of the Rossija ribotype along with other rare ribotypes associated with non-toxigenic strains (Cluj and Rossija, in particular).

Molecular detection of fluoroquinolone resistance-associated gyrA mutations in ofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Iran and Belarus.

OBJECTIVE/BACKGROUND: Detection of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene could determine resistance to fluoroquinolone antituberculosis drugs. The aim of this study was to detect mutations in QRDRs. METHODS: From 184 clinical isolates of Mycobacterium tuberculosis, ofloxacin resistance was proven in 42 isolates using the proportion method. The molecular basis of resistance to ofloxacin were investigated by the determination of mutations in the QRDR region of the gyrA gene. Extracted DNA fragments of 194bp from the gyrA gene were amplified and an automatic DNA sequencer was used for the sequencing process. RESULTS: Molecular genetic analysis of 42 resistant M. tuberculosis strains demonstrated that they belong to Principal Genetic Group (PGG) 1 in 19 cases (45.2±10.9%), to PGG2 in 15 cases (35.7±10.5%), and to PGG3 in eight cases (19.0±8.4%). Isolates from PGG1 were dominant among resistant isolates (P<.05). It was found that 24 (57%) resistant isolates carried mutations at codon 94 with five different amino acid changes: D94A (n=11), D94G (n=3), D94T (n=4), D94A (n=4), and D94Y (n=2). The remaining 18 (43%) isolates had mutations in codon A90V (GCG→GTG) and S91P (TCG→CCG). Five isolates had two mutations in codons 90 and 94. There was no difference between mutations at these two codons in resistant isolates of the two countries (P<.001). There was no polymorphism observed in codon 95 in any of the ofloxacin-susceptible isolates. CONCLUSION: It was concluded that the determination of nucleotide sequences of QRDRs can be used as a molecular test for the rapid detection of ofloxacin resistance. Furthermore, frequencies in gyrA codons in Belarus and Iran were similar, therefore it is not of geographical concern for the two countries.

Antibiotic policies in Central Eastern Europe.

To assess the antibiotic policies in Central Eastern European (CEE) countries, a questionnaire on the prevalence of resistance, antibiotic consumption data for ambulatory and hospital care and antibiotic policies, was mailed to national representatives. Data on antibiotic resistance and consumption of antibiotics at national levels are limited and vary considerably among countries. The importance of surveillance data in altering perceptions of the prevalence of resistance is shown by the comparison of surveillance data and interview data. Interview data without surveillance data produced the widest range of estimates of the prevalence of resistance in streptococcus pneumonia -5% in Lithuania and 82% in Belarus. The average consumption of antibiotics in ambulatory care in eight CEE countries in 2001 was 19.35 defined daily doses (DDD)/1000 inhabitants per day, (range 13.1 - 24.8 DDD) and in hospitals in six CEE countries was 2.2 DDD/1000 inhabitants per day (range 1.3-4.5). Over the counter sales of antibiotics are available in some countries. Antibiotic policy interventions do not exist or only apply to specific problems or interventions. Better implementation of antibiotic interventions and education on antibiotic use should be a high priority in this region. An effective strategy requires close co-operation, consultations and partnership at national and international level in particular, via existing international organisations.